Avian Influenza Summary

• Worldwide distribution

• Sporadic outbreaks of High Path H5 and H7 in poultry

• Wild aquatic birds are reservoirs for all avian influenza viruses

• Viruses can change quickly (mutation)

• Avian-origin influenza viruses contribute to outbreaks in humans and other animals

• Virus is inactivated by most disinfectants

• Vaccination not done in the US, but is done in countries with HPAI

• Good biosecurity is effective prevention

• Sudden onset, moderate to high

morbidity and mortality.

• Edema of head

• Cyanosis/necrosis of comb/wattle

• Subcutaneous hemorrhage

HP Avian Influenza

Classic Signs & Lesions:

Unique Features

• Unprecedented spread to three continents

• Unprecedented number and variety of

species affected

– Many avian and multiple mammalian

species are susceptible

• Spill-over to wild birds, causing mortality in

wild waterfowl

• Involvement of waterfowl in virus spread

• Unprecedented changes in the virus

• High mortality in humans

HPAI H5N1 Outbreak

HPAI H5N1 Outbreak

Jan 2009. http://www.pandemicflu.gov/

>250 million chickens died/killed

>60 Countries affected (27 in 2008)

391 confirmed human cases (15

countries) -- 247 fatalities

All HPAI Outbreaks from Jan 2011 to Jan 2012

Avian Influenza Virus

HA

PB1

PB2

PA

NP

NAMA

NS

M2

Hemagglutinin

Neuraminidase

M1

Orthomyxovirus • Type A

•Humans are susceptible to A, B, and C

• Enveloped virus

• RNA

• Single stranded (negative sense)

•8 segments, codes 10

proteins

• Major surface proteins

• Hemagglutinin (H1-H16)

• Neuraminidase (N1-N9)

• 144 potential combinations

• H5N1, H7N7, H9N2 etc.

Avian Influenza Viruses Change Frequently

• Antigenic Drift: mutations in H gene cause minor antigenic changes to H protein

– Continuous process

– Limits protective immunity

• Antigenic Shift: Replacement of H or N

– Genetic re-assortment (human and animal viruses)

– Insertion of large section of extraneous RNA near the HA cleavage site – virulence shift

– Emergence of novel (pandemic) strains

Natural Reservoirs of Influenza A Viruses

• Wild aquatic birds

• Majority of influenza isolates comes from two orders:

1. Anseriformes: ducks, geese, swans

-Mallards and other dabbling ducks have highest isolation rates.

2. Charadriiformes: gulls, terns, and shorebirds such as ruddy turnstones

• Typically, no clinical disease except: 1. South Africa in 1961

2. H5N1 infection in wild birds in Asia

Ecology of Influenza A

Genetic

Reservoirs

Intermixing

H1, H3

H1, H2, H3 H3, H7

Commercial, LBMs Others H1-12

H14-15 H1-2, 4-7, H9-13, 15-16

H10

H1, H3, H4, H7, H13

Other Aquatic Birds?

H5N1

Modified from D. Swayne

H3

H1N1

Emergence of HPAI

Reservoir

Genetically stable

and low path in

natural host Most

HPAI

Asian

HPAI H5N1

Mutation- H5/7

may evolve to HP

Exposure/adaptation

to domestic species

that are not natural

hosts

Highly Pathogenic (HPAI)

Low Pathogenicity (LPAI)

Classification of Avian Influenza

• Localized infection

• No to mild disease

• Any subtype (H1-H16)

• Waterfowl (gut)

• Domestic birds (resp)

• LP H5 and H7 in poultry

are reported to OIE

• Acute, systemic disease

• High mortality

• H5 & H7 subtypes only

• Affects primarily poultry

(chickens, turkey, etc)

• Reported to OIE

Avian Influenza

Factors Influencing Pathogenicity:

• Only H5 or H7 subtypes are HP, but the majority of H5 and H7 are LP

• Polygenic trait, but HA plays dominant role

• Multiple basic amino acids at HA cleavage site

• Glycosylation sites near HA cleavage site

Remember: pathogenicity in the field is highly variable depending not only on the virus, but also the species and age of bird infected, concurrent infections, and other factors

1. Any influenza virus that kills 6, 7, or 8 of 8

chickens (75% mortality) or IVPI>1.2

2. Any H5 or H7 subtype that does not meet

the criteria in item 1, but has an amino acid

sequence at the cleavage site of the

hemagglutinin that is compatible with other

HPAI viruses

Note: The OIE also is notified when low path H5

and H7 is found in domestic poultry.

Highly Pathogenic Avian Influenza USDA/OIE definition:

Transmission Sources of virus Routes of transmission

Major

• Infected poultry flocks and

live bird markets

• Feces/resp secretions

• Infected wild waterfowl

Minor

• Uncooked infected poultry

products

• H1/H3 from swine to

turkeys

• Pet birds

• Oral - feces or cannibalism

• Aerosol

• IN, IC, IP, IM, IV, IC

• Fomites

– People

– Equipment

– Vehicles

– Feed and water

– Rodents, sparrows, etc

– Wind (less than 0.5mi)

Incubation and shedding of virus

• Incubation period of hours to 3 days

• Duration of virus shedding is highly variable

• More important than individual shedding is

flock shedding of virus

– AI viruses can be maintained for long

periods in domestic and wild flocks via

individual, susceptible birds

Days at Condition

Stability of Hong Kong H5N1 Virus in Feces at Environmental Temperatures

Shortridge et al. 1998

0 1 2 3 4 5 8 17 21 28 40 0

1

2

3

4

5

Vir

us

Tit

er

(lo

g 1

0)

Wet Feces

(25 C)

Dry Feces

(25 C)

Wet Feces

(35 C)

Wet Feces

(4 C)

Common Findings with Low Path AI

• Flocks with respiratory disease

• Flocks with decreased egg production

• Flocks with decreased food/water intake

• Increased daily mortality >3 bird deaths/1000 for 2

days

• These characteristics are not specific to AI

– Many possible differential diagnoses at this

stage

• These signs may only be identified retrospectively

after routine serologic surveillance detects LPAI

LP Avian Influenza

I.

Capua

D. Halvorson

•Coughing, sneezing, rales

•Sinusitis, tracheitis, air sacculitis

•Bronchopneumonia w/ secondary

pathogens (P. mult, E. coli)

•Epiphora

•Depression and huddling

•Ruffled feathers

D. Halvorson

LP Avian Influenza

• Rough, misshapen eggs

• Hemorrhage in ovary

• Oviduct edema

• Egg yolk peritonitis

• Catarrhal enteritis

• Urates in kidney

D. Swayne D. Swayne

Common Findings with HPAI • Sudden onset and rapid spread

• Mod to high morbidity and

mortality (>20 dead/1000/day)

• Sudden death

• Severe depression, drop in

feed/water consumption

• Cough, sneeze, nasal discharge

• Ataxia, tremors, torticollis

• Dramatic decreased egg

production and abnormal eggs

HPAI

HPAI

Dr. H K Wong, Hong Kong Zoo

HPAI

Dr. A. Wilson, NVSL Asian H5N1

HPAI

HPAI

HPAI

HPAI

HPAI Differential diagnosis

• Infectious laryngotracheitis (ILT)

• Infectious bronchitis (IB)

• Fowl cholera (P. multocida)

• Mycoplasma gallisepticum

Acute high mortality

• Newcastle disease

• Water deprivation

• Heat exhaustion

• Toxins

Respiratory disease

Sample collection

1. Serology (Important diagnostic specimen)

• AGID, ELISA (type-specific), and HI/NI

(subtype-specific)

– 0.5 to 1 ml of serum, plasma, egg yolk

– 4°C or minus 20°C

– Typically sample 30 birds per flock

+

-

+

AG

AS AS

AS

Sample collection for VI or rRT-PCR

2. Tissue

– Lung and spleen

– Ship samples in plastic bag, and then double bagged

– Do not pool tissues from different birds

– Typically sample 3-5 sick birds per flock

3. Swabs: oropharyngeal (OP) and cloacal (CL)

– Use sterile polyester swabs on plastic shafts

– Poultry: can pool 5/tube, but do not mix OP with CL swabs

– Water fowl use individual cloacal swabs

– Place swabs in 2-4ml brain-heart infusion (BHI) broth

• Shipping/Storage of tissue and swabs

– Short term: 4°C (up to 72 hours)

– Long term: minus 70°C (minus 20°C not recommended)

Virus Isolation

• Embryonating chicken eggs

• Expensive and labor intensive

• Special facilities (BSL-2, -3Ag)

• Test all sample types

• Sensitivity – high (all subtypes), may have low

sensitivity to some wild bird influenza viruses

• Specificity – low (grow many viruses, bacteria)

– Detection of AI is by HA

– Differentiation from other HA agents by HI

e – 2 days-2 weeks

Diagnostics

Real Time RT-PCR (rRT-PCR) • Target

– Influenza A virus (matrix gene)

– H5, H7, N1 subtypes validated

• Moderate cost/test ($8)

• Expensive equipment ($25-90K)

• Sensitivity – high (similar to VI)

(assuming using right primers)

• Specificity – high, Time - rapid (3-5 hrs)

• Potential for high throughput

• Live virus not required

Diagnostics

Antigen Capture Immunoassays

• Commercially available (several sources)

• Detection of influenza A virus

• Pen-side – no special facilities required

• Sensitivity – low/moderate (70-80% compared to

VI, need 3-5 logs of virus)

– Good for testing sick or dead birds

– Low sensitivity for vaccinated populations,

healthy birds, and live wild bird surveillance

• Specificity – high

• Moderate cost ($7-8) and rapid (10-15 minutes)

Synbiotics – Flu Detect Test

Diagnostics

AI Control • Strategies:

–Prevention – prevent introduction of AIV

–Control – reduce economic impact – limit spread

–Eradication – total elimination of AIV

• Achieved through…

–Biosecurity (exclusion and containment)

–Diagnostics and surveillance – early detection

–Elimination of AI virus in infected poultry

–Reduce susceptibility through vaccination

–Education of producers and public

National Biosecurity • Import restrictions on poultry and poultry

products countries with HPAI

• Heighten border surveillance for illegally

imported poultry and poultry products

• Wild bird surveillance programs

– 2006 (>160,000 specimens)

– 2007 (>95,000 specimens)

– 2008 (>50,000 specimens)

– 2009 (>19,000 specimens)

• Imported live birds

– 30-day quarantine

– Laboratory testing for AIV

Van Borm S, EID 2005

Farm Biosecurity • Avian influenza is a preventable disease

• Protect poultry, feed and water from contact with

reservoir hosts/feces

• All-in, all-out management ideally, with a single species

and single age group

• Purchase birds from reputable, tested supplier and/or

quarantine and test incoming birds

• Limit access to farm

• Provide clean clothing and boots to workers

• Clean and decontaminate all vehicles and equipment

coming on to the farm

• Don’t bring in equipment from other farms

• Avoid visiting other farms and live bird markets

Personal Biosecurity for HPAI

• Educate staff on disinfection, hygiene, etc.

• Disposable nitrile or vinyl gloves

• Disposable coverall with impermeable apron

• Shoe covers or boots, which can be disinfected

• Safety goggles

• Respirator (N-95, N-99, N-100), requires fit testing

• Prophylactic antiviral medications

• Seasonal flu vax and medical surveillance of workers

AI PPE recommendations from the CDC http://www.cdc.gov/flu/avian/professional/protect-

guid.htm See website for complete details

Iodophores

Phenolics

Quaternary ammoniums

Ethanol

Bleach

Peroxygens (Virkon-S)

Food Safety

• Eating properly handled and cooked

poultry and eggs is safe

• The chance of HPAI infected meat getting

into the human food chain in the US is

extremely low

• Wash hands with warm, soapy water for 20

seconds

• Wash utensils with hot, soapy water

• Sanitize cutting boards with dilute bleach

• Cook poultry to an internal temperature of

165°F

Control and Eradication

• Vaccination – not a stand alone control method

– Advantages • Reduce viral shedding

• Prevent illness and death (does not prevent infection)

– Disadvantages • No cross protection between subtypes

• Can interfere with diagnostics and surveillance

• Can hide clinical signs

• Impairment to international trade unless DIVA is used

• Vaccine bank

US Surveillance Programs Federal, State and Private laboratories

• Diagnostic surveillance

– Unexplained respiratory disease, decreased

egg production, and increased mortality

– FADD investigations

• Live bird market surveillance

• Commercial producers surveillance via NPIP

and national H5 and H7 LPAI control program

• Wild Migratory Bird surveillance